Saponins, glycosides widely distributed in the plant kingdom, include diverse ring compounds formed by the non-sugar portion thereof. Triterpene saponin is a saponin that is contained in ginseng or red ginseng as a major physiologically active ingredient, and this ginseng saponin is named ginsenoside, which means ginseng glycoside, to specifically distinguish it from other vegetables' saponin based on a different chemical structure.
Ginsenosides are classified into three groups based on their aglycone structure: Protopanaxadiol-type ginsenosides, Protopanaxatriol-type ginsenosides, and Oleanolic acid-type ginsenosides. These three groups are further classified based on the position and number of sugar moieties (aglycones) attached by a glycosidic bond at the C-3, C-6, and C-20 positions of the rings in the chemical structure. The oleanolic acid-type ginsenoside has a pentacyclic backbone and ginsenoside Ro is the only saponin having oleanolic acid as aglycone. To date, more than 40 ginsenosides have been isolated, and most of them are Protopanaxadiol-type ginsenosides. Protopanaxadiol-type ginsenosides include Rb1, Rb2, Rb3, Rc, Rd, Gypenoside XVII, Compound O, Compound Mc1, F2, Compound Y, Compound Mc, Rg3, Rh2, and C-K.
In addition, major ginsenosides account for over 90% of total ginsenoside content, but show a very low in vivo absorption because of their large size. Therefore, in order to increase the efficacy of ginsenoside, it is required that major ginsenosides are converted into rare ginsenosides showing a relatively excellent absorption and efficacy. That is, deglycosylation of major ginsenosides is required to show effective physiological activities in vivo (Tawab et al., 2003; Akao et al., 1998). The major ginsenosides include Rg1, Re, Rb1, Rc, Rb2, or the like, and the rare ginsenosides include F2, Rg3, Rh1, Rh2 and compound K, C-K, Mc, Mc1 or the like.
Among the rare ginsenosides, Mc1 is a ginsenoside that is mainly included in red ginseng in a trace amount, and the compound Mc1 is produced as a metabolite upon intake of diol-type ginsenoside Rc having functions of alleviating pain, accelerating secretion of corticosterone, suppressing hypertrophy of glomerulus, suppressing lipid peroxidation of liver cell, and stimulating the synthesis of bone marrow cell, DNA, RNA, protein, and lipid. Various studies on the pharmaceutical activities of the compound Mc1 have not been made yet, because it is present in a very small amount.
Further, rare ginsenoside F2 has been known as an ingredient having effects of suppressing proliferation of tumor cells and reversing the multi-drug resistance in tumor cells or bacteria (Korean Journal of Pharmacognosy 28(1), 35, Jong Hwan Sung et al., 1997). Ginsenoside F2 is found in only small amounts in ginseng. When administered in the body, saponin is finally transformed into compound K via the intermediate metabolite, ginsenoside F2. Therefore, it is very difficult to produce a large amount of ginsenoside F2.
Meanwhile, the known preparation method of ginsenoside F2 is a method of preparing ginsenoside F2 by hydrolysis of a diol-based saponin using an enzyme naringinase or by the decomposition in the rat large intestine after oral administration. However, these methods show very low yields, and it is difficult to produce high-purity ginsenoside F2 due to the production of various secondary metabolites (Koizumi et al., Chem. Pharm. Bull. 30(7):2393, 1982; Karikura et al., Chem. Pharm. Bull.). Even though the mass-production methods thereof have been studied, effective methods have not yet been established.
Further, ginsenoside F2 has been known to have the effects of suppressing the proliferation of tumor cells and reversing multi-drug resistance in tumor cells or bacteria (Korean Journal of Pharmacognosy 28(1), 35, Jong Hwan Sung et al., 1997). It is known that ginseng ginsenosides are metabolized by intestinal flora such as Prevotella Oris after they are orally administered, and their metabolite F2 shows pharmaceutical effects. However, the useful F2 is also present in only small amounts in some ginsengs, and thus it is difficult to produce a large amount thereof. In addition, it is difficult to produce only high-purity F2 because of the production of various secondary metabolites during the metabolic process.
For the production of rare ginsenosides present in a small amount, chemical decomposition (De Mayo et al., canad. J. Chem., 43, 2033, 1965), an enzymatic method (Kitagawa et al., Terahedron Letters, 30, 2283, 1974), and glycoside synthesis (Korean Patent No. 10-2005-0007250) have been suggested, but these methods have limitations in mass-production such as 1) requiring many production steps for the production process, 2) loss of desired compounds during processing, 3) use of inedible catalysts, or 4) low yield.
In particular, enzymes such as β-glucosidase, β-L-arabinopyranosidase, β-L-arabinofuranosidase, and β-L-rhamnosidase can be used in the enzymatic method, and there have been many studies on biotransformation of major ginsenosides using these enzymes. However, these methods are also not effective for mass-production, and have a problem in that they require high production costs.
Moreover, not all enzymes of β-glucosidase, β-L-arabinopyranosidase, β-L-arabinofuranosidase, and β-L-rhamnosidase do have the activity of biotransformation of major ginsenosides into rare ginsenosides. For example, the present inventors demonstrated that beta-glucosidase known to be derived from Arthrobacter chlorophenolicus A6 has no activity of the biotransformation of ginsenoside. In addition, even though a known enzyme, for example, beta-glucosidase A, is known to have biotransformation activity into Rb1, the activity was not satisfactory.
Korean Patent Application No. 10-1999-0045180 provides a method for preparing ginsenoside Rh2 by degradation of saccharide of PPD-type ginsenoside using saponin alpha-glucosidase. The saponin alpha-glucosidase of the above invention converts ginsenoside Rd into ginsenoside Rh2 via ginsenoside F2. In addition, ginsenoside Rh2 can be produced from ginsenoside Rb1 and Rc. As described in Example 3, however, the saponin alpha-glucosidase should be obtained by removal of bacterial cells from the culture broth of Aspergillus in media containing wheat bran and ginseng powder. Thus, the resulting low production yield increases the production cost in mass-production, and loss or the desired product problematically occurs during the production process.